A key biochemical change observed in diabetes mellitus in humans and in animal models of the disease is an increase in the chemcial attachment of glucose to proteins which occurs without the aid of enzymes (N nonenzymatic glycation," formerly called "nonenzymatic glycosylation"). This occurs due to the increased glucose concentration in the blood of poorly-controlled diabetics. Therefore, the amount of nonenzymatic glycation of a given protein is indicative of how well a diabetic is controlling his or her blood glucose concentration, and may also be of value in predicting the progression of tissue complications that occur in diabetes such as renal, ocular, microvaascular and nervous system disease.
Many proteins such as hemoglobin, albunim, fibrinogen, fibrin, low density lipoproteins (LDL), lens crystallins, peripheral nerve proteins, interstitial collagens and type IV basement membrane collagen, have been found to be nonenzymatically glycated to a greater extent in diabetic patients than in normal subjects. The glycation reaction results in the attachment of glucose to proteins via nucleophilic addition to form a Schiff base between glucose and the N-terminal amino group of apolypeptide or the epsilon-amino group of a lysine residue in the polypeptide chain. The formation of the initial linkage (the labile glucose adduct formed via an aldimine linkage) is reversible. Therefore, the Schiff base reaches an equilibrium level in vivo which reflects the ambient glucose concentrations. With time, however, there is a slow chemical rearrangement of the Schiff base (termed an Amadori rearrangement") which results in the formation of a stable ketoamine (the 1-amino-1-deoxy-2-keto adduct termed the Amadori product"). The kinetics of these reactions have been documented by studies of the steps involved in the formation of the glycated Amadori product, hemoglobin A.sub. 1c.
In terms of the tissue complications of diabetes mellitus, the nonenzymatic glycation of various proteins has been implicated in a number of pathological sequelae, including the progression of kidney disease, cataract formation, neuropathy, and atherosclerosis. For example, glycation of hemoglobin alters its affinity for oxygen. Lens crystallin glycation results in opacification and may contribute to cataract formation. Glycation of collagen alters the extent and perhaps the type of collagen cross-linking that leads to stiffening of tissues. Glycation of LDL alters cellular uptake and degradation of this protein. The nonenzymatic glycation of fibronectin, laminin and type IV collagen alters the molecular association of these molecules with each other and with heparan sulfate proteoglycan, and may alter the composition of basement membranes in tissues affected by the complications of diabetes.
The development of quantitative methods for the measurement of the nonenzymatic glycation of proteins has been carried out mainly on hemoglobin. Because of the relatively long half-life of red blood cells (approximately 60 days), when properly done, the glycosylated hemoglobin assay provides a retrospective index of glucose control in patients that correlates well with mean plasma glucose levels, 24-hour urinary glucose concentrations, and other indexes of metabolic control determined over the preceding two to three months.
However, the quantitation of glycation levels in proteins other than hemoglobin is important since other readily accessible proteins in plasma, urine, or tissue biopsies can provide information about glycemic control within different time frames. For example, the half-life of albumin or low density lipoproteins is 3 to 5 days and the measurement of the glycation of these proteins may indicate the degree of glucose control over a very short period of time. On the other hand, the quantitation of glycation levels of skin collagen (half-life of approximately 2-3 years), for example, would indicate the ability of diabetic subjects to regulate glucose concentrations over a much longer period of time than that which can be determined by measuring glycated hemoglobin.
Assays have been designed to measure the total glycation levels of the adult form of hemoglobin (hemoglobin A). This glycated fraction of hemoglobin A (termed "hemoglobin A.sub.1 " is modified by glucose at .beta.-chain terminal valine residues and at epsilon-amine groups of internal lysine residues and is more negatively charged than normal hemoglobin A (unmodified hemoglobin or hemoglobin A.sub. 0). Hemoglobin A.sub. 1c, another clinically useful substrate for the measurement of nonenzymatic glycation, on the other hand, is a subfraction of hemoglobin A.sub. 1 which consists of hemoglobin A glycated by a ketoamine linkage at only the .beta.-chain terminal valine residue.
Immunological approaches to the measurement of the levels of nonenzymatic glycation of hemoglobin have been attempted using .sup.125 I-labelled antibody in a radioimmunoassay. The first of these approaches is based on the observation that glycated products cannot be demonstrated in sheep red cell hemolysates. This may be because sheep hemoglobin lacks the "diphosphoglycerate pocket" which permits the glycation of the .beta.-chain N-terminus of hemoglobin. The sheep, as disclosed by J. Javid, et al., Brit. J. Haematology, 38, 329 (1978), therefore, recognizes the N-terminus of human hemoglobin A.sub. 1c as foreign and produces an antibody against it. However, this polyclonal antibody is difficult to raise and it also cross-reacts with hemoglobin A.sub. 1a and hemoglobin A.sub. 1b, chromatographically-stable components of hemoglobin A.sub. 1 which are distinct from the A.sub. 1c species. The A.sub. 1c antisera must also be repeatedly absorbed with agarose-linked hemoglobin A.sub. 0 at the expense of a considerable loss of antibody titer. The observation that the antibody to human A.sub. 1c reacts less well with dog and mouse hemoglobin A.sub. 1c also raises the possibility that the steric fit of the antibody includes more than the sugar molecule and probably extends to surface features of the protein adjacent to the glucose modification.
L.K. Curtiss, et al., in J. Clin. Invest., 72, 1427 (1983) have disclosed the formation of murine monoclonal antibodies which react with nonenzymatically-glycated murine low density lipoprotein. However, the glucose adducts on the protein had to be first chemically reduced with sodium borohydride or sodium cyanoborohydride to yield an immunogenic hexose alcohol (glucitol-lysine) since these authors did not succeed in raising monoclonal antibodies to the unreduced adducts naturally found on proteins in diabetic tissues (the labile Schiff's base or Amadori product). It would, therefore, also be necessary to reduce the target proteins in a test sample in a similar fashion to produce glucitol-lysine residues in order to obtain reaction with the antibody. The clinical utility of this method remains to be determined, especially since the detection of various glycated epsilon-amino groups of lysine on a protein is limited to those that can be selectively reduced by chemical-reducing agents in vitro.
Therefore, a need exists for monoclonal antibodies which recognize and will selectively react with the unmodified Schiff's base or Amadori glucose adducts which result from the nonenzymatic glycation of proteins, such as the proteins associated with physiological fluids such as blood and lymph.